Use of glucose-6-phosphate isomerase and antibodies thereto for the diagnosis and therapy of arthritis, and test of anti-arthritic compounds

ABSTRACT

The present invention relates to the use of antibodies against glucose-6-phosphate isomerase (GPI) and like protein for diagnosis of arthritis and the use of said protein for treatment of arthritis. It is also aimed at a process for isolating monoclonal antibodies capable of transferring arthritis and antibodies thereof, as well as a method for determining the anti-arthritis potential of a composition.

The application is a Divisional of application Ser. No. 09/959,230 filedOct. 22, 2001, issued as U.S. Pat. No. 7,033,745.

The present invention relates to the use of antibodies againstglucose-6-phosphate isomerase and like protein for diagnosis ofarthritis and the use of said protein for treatment of arthritis.

Rheumatoid arthritis (RA) is a frequent and incapacitating autoimmunedisorder (Feldmann et al. 1996). It is a chronic, progressive jointdisease, characterized by leukocyte invasion of the synovial lining andhyperplasia of the resident synoviocytes. The ensuing overproduction ofcytokines and other soluble mediators results in cartilage destruction,bone erosion and anarchic remodelling of joint structures. The etiologyand pathogenesis of RA remain controversial. It is not known whetherdisease is initiated by an unrestrained inflammatory reaction to amicrobial antigen (Ag), an inappropriate autoimmune response to aself-constituent, or both. An important role for T cells (Panayi et al.,1992), B cells (Zvaifler, 1973), and other leukocytes such as dendriticcells, macrophages and neutrophils (Thomas and Lipsky, 1996) has beenargued, and disputed. The lack of a consensus largely reflects twofactors. RA is a heterogeneous syndrome, different patients exhibitingwidely variant ages of onset, disease courses, genetic profiles andresponses to therapeutic intervention. In addition, there has been adearth of small animal models of RA, particularly those spontaneouslysuccumbing to disease.

Recently, we generated a new transgenic mouse model of arthritis thatspontaneously develops a disease with many of the characteristics ofrheumatoid arthritis in humans ((Kouskoff et al., 1996), U.S. Pat. No.5,675,060). All KRN T cell receptor (TCR) transgenic (tg) mice on theC57Bl/6xNOD genetic background (hereafter abbreviated as K/BxN mice)develop a joint disorder, starting at three to four weeks of age, andrapidly evolving until the animal's mobility is severely compromised; asin patients, the disease is chronic, progressive, symmetrical and has aproximal to distal gradient of severity. The murine disease exhibits allof the major histological features of the human one: leukocyte invasion,synovitis, pannus formation, cartilage and bone destruction, anarchicremodelling. The mouse model, like patients, shows several immunologicalabnormalities, including B cell hyperactivity manifest as an increase inB cell numbers, hypergammaglobulinemia and autoantibody production.

The disease in K/BxN mice is initiated by cross-reactive recognition ofNOD-derived A^(g7) molecules of the Major Histocompatibility Complex bythe KRN TCR. Thus, in K/BxN animals, a situation of systemicautoreactivity is generated, raising the issue of how joint-specificautoimmune disease develops in the presence of systemic autoreactivity.We have reported earlier that T lymphocytes are required for arthritisdevelopment, as their blockade of T lymphocytes prevents disease,although they appear dispensable at the later stages of disease. Blymphocytes are also critical (Kouskoff et al., 1996).

In the present invention, we find that B cells are required forspontaneous arthritis because they produce pathogenic immunoglobulindirected against a target we identify as glucose-6-phosphate isomerase(EC 5.3.1.9). We show that recombinant glucose-6-phosphate isomerase canbe used in diagnostic tests of murine arthritis, and to adsorbpathogenic immunoglobulins.

Autoantibodies have been previously reported in situations of inducedarthritis in animal models, when disease is induced by immunization withcartilage components, such as type II collagen. Antibodies againstubiquitously expressed proteins have not been previously found to bearthritogenic.

Autoantibodies are also found in serum from RA patients, and have somediagnostic value. Rheumatoid factor (RF; Ab against the Fc portion ofIgG) has been considered a hallmark of this disease; however, it isabsent from about 30% of RA patients and is present in individuals withother autoimmune diseases (Mannik, 1992; Rudolphi et al., 1997) or insituation of chronic immune stimulation. Abs against cartilage orepidermal components have also been detected in RA patients, directedagainst Type II collagen (cII) or filaggrin, for example, but, again,these generally show limited correlation with disease parameters(Rudolphi et al., 1997; Claque and Moore, 1984; Sebbag et al., 1995).

The present invention deals with antibodies against glucose-6-phosphateisomerase (GPI) and glucose-6-phosphate isomerase like proteins (GPIlike).

Said antibodies may be polyclonal or monoclonal and the technologiesused for their preparation are known by the man skilled in the art.

GPI like proteins are proteins found in the serum, antibodies againstwhom have arthritogenic potential. Said GPI like are generally expressedin many tissues but always released in the serum, but are unable toinduce complete immunological tolerance, and are thus the target ofautoreactive T lymphocytes and/or autoantibodies.

GPI like proteins may also be proteins or polypeptides which contain atleast one epitope able to interfere with anti-GPI or anti-GPI likeantibodies, especially autoantibodies. Such proteins or polypeptides maybe synthetic or recombinant and may encompass sequences of otherproteins, especially said proteins or polypeptides when used fortherapeutic application are not able to induce any pathology.

Examples of the present invention give the man skilled in the artdetailed indications on how to identify GPI like proteins. In thisregard, the present invention provides a test to isolate GPI likeproteins and determine their arthritogenic property. One possibleroutine procedure is:

-   -   obtaining serum from arthritic mammals, such as the K/BxN        transgenic mice,    -   preparing different IgG fractions of said serum and testing them        for their ability to induce arthritis and eventually preparing        subfractions based on these fractions,    -   preparing an affinity chromatography column with IgG of said        fractions,    -   isolating and purifying GPI like proteins from proteins extracts        from organs of the same mammal by means of said column.

These isolated proteins, defined as GPI like proteins, can be furthercharacterized by means of standard techniques such as sequencing.Technical details on the experiments performed that led to theidentification of GPI are described in Matsumoto I. et al (1999) andKorganow A S. et al (1999), incorporated herein by reference.

Therefore, another aspect of the invention is directed to a process forisolating GPI like proteins comprising the steps consisting of:

-   -   preparing different IgG fractions or subfractions from serum of        arthritic mammals, preferably from the K/BxN transgenic mice,    -   isolating GPI like proteins by affinity chromatography with IgG        of said fractions as matrix.

Said GPI like proteins are essentially enzymes, for example an enzymeinvolved in the hexose metabolism pathway, preferably GPI analogs.

The invention also concerns the use of the GPI or GPI like proteins andantibodies thereto obtainable by the process as depicted above for thetreatment and diagnosis of arthritis.

Antibodies according the invention are different from the knownarthritogenic antibodies described in the prior art which are directedagainst cartilage components such a Type II collagen. The antibodies ofthe present invention are mainly directed against proteins, which areexpressed in many non-articular tissues, but released in the serum.

Another embodiment of the present invention deals with a process fordiagnosis of arthritis which comprises detection of autoantibodiesagainst GPI or GPI like proteins in the sample of plasma or serum of apatient.

The method of testing for the presence or amount of antibodies againstGPI or GPI like proteins present in a sample comprises binding saidproteins to the antibodies in the sample and detecting antibodies boundto said proteins.

The processes for detection of antibodies in the plasma or serum arewell known in the art, for example ELISA or RIA tests may be used. It ispossible to use a substrate coated with GPI or GPI like proteins, forexample an assay plate, and then detect the presence of antibodies fixedon said GPI with appropriate labeled antibodies. Among the labels, thosewho are preferred are radioactive isotopes, compounds containing anisotope, enzymes, in particular enzymes susceptible to react withchromogenes, fluorigenes or luminescents (for example a peroxydase or analcaline phosphatase), chromophores, chromogene compounds, fluorigenesou luminescents, base analogues, and ligands such as biotin.

According to the present invention, GPI or GPI like proteins may be thenatural protein itself, recombinant protein, synthetic protein or onlythe recombinant epitope of said proteins which are also encompassed bythe terminology GPI like. So, the present invention also deals with akit for carrying out the diagnosis of antibodies against GPI or GPI likeproteins comprising a diagnostic agent consisting of a protein which isGPI or GPI like or an epitope thereof capable of interacting with anautoantibody of the plasma or the serum and a second antibody able tobind to said autoantibody, said second antibody being labelled fordetection.

In another embodiment, the present invention concerns the use of GPI orGPI like proteins including portion of said GPI like which are able tointeract with autoantibodies to inhibit or remove said autoantibodiesfor treatment or prevention of arthritis.

The invention also deals with the expression of GPI or GPI like proteinsin vivo through the use of a vector comprising elements sufficient forexpressing in vivo said GPI or GPI like proteins. Said vector may befrom viral origin, may be plasmid or may be purely synthetic or nakedDNA as described in VICAL technology. Expression of said proteins willprovide GPI like proteins to adsorb arthritogenic antibodies (see theexamples of said specification).

The efficacy of potential anti-arthritic compositions can be evaluatedby determining the effect of the compositions on arthritis induced innormal mice by injection of serum from K/BxN mice, or of immunoglobulinsderived therefrom, or of anti-GPI or anti-GPI like antibodies.Compositions thus evaluated include, but are not limited to, chemicalcompounds, biologicals such as antibodies, polypeptides,anti-inflammatory agents, hormones, tolerogens, which inhibit thebinding of anti-GPI or anti-GPI like antibodies to their targets or thepathological consequences of this binding.

This can be done by: i) administering a known dose of arthritogenicserum or immunoglobulins to a first non-arthritic mouse, said mouse alsoreceiving the arthritogenic composition to be tested, administeredeither before, together, or after the arthritogenic serum orimmunoglobulins; ii) detecting the time course of inflammation and jointdestruction in said first mouse; and iii) comparing the time course ofinflammation and joint destruction in said first mouse to the timecourse of inflamation and joint destruction in a second mouse of samegenotype receiving the same arthritogenic serum or immunoglobulins butwhich has not been exposed to said anti-arthritic composition.

Another possibility for step ii) is to detect the extent ofinflammation, joint destruction and limb deformation in said firstmouse; and for step iii) to compare the extent of inflammation, jointdestruction and limb deformation in said first mouse to the extent ofinflamation, joint destruction and limb deformation in a second mouse ofsame genotype receiving the same arthritogenic serum or immunoglobulinsbut which has not been exposed to said anti-arthritic composition.

In this process, the arthritogenic immunoglobulins can be monoclonalantibodies or combinations thereof, especially antibodies describedbelow.

It is also possible to test molecules which are able to modulate theproduction of GPI or GPI like proteins in order to treat arthritis, saidmolecule may be for example antibodies which are non pathogenic.

It is also possible to use surface coated with GPI or GPI like proteinsfor treatment of serum in an extracorporeal circuit for depleting thequantities of pathological antibodies.

Another aspect of the invention relates to a process for isolatingmonoclonal antibodies capable of transferring arthritis comprising thesteps consisting of:

-   a) preparing hybridomas from lymphocytes of arthritic mammals    splenocytes, preferably from K/BxN mice splenocytes,-   b) selecting said hybridomas in limited dilution conditions in HAT    medium,-   c) screening hybridomas producing antibodies directed against GPI or    GPI like proteins in an ELISA assay,-   d) expansion and cloning of the selected hybridromas at step c),-   e) testing said antibodies produced by selected hybridomas at    step d) in combination or alone for their ability to transfer    arthritis.

The testing of step e) can be achieved by i): administering a known doseof said antibodies to a normal mouse; ii) detecting the onset ofinflammation joint destruction and limb deformation in said mouse.

It is to be understood that the above process steps can be achieved withwell known techniques pertaining to the art and are not limited to anyparticular procedure.

So, the invention is directed to monoclonal antibodies obtainable bysuch a process and to hybridoma lines producing said antibodies. Amongthe antibodies depicted at example 5, antibodies 2.99 and 6.121 arepreferred (see FIG. 9 below). These antibodies can be used to the sameends as the serum or anti-GPI immunoglobulins described above.

Some additional characteristics and advantages of this invention willappear from the following examples which will be explain in reference tothe figures wherein:

FIG. 1. Serum from K/BxN mice can transfer arthritis.

A: Paws of normal mice injected 72 h previously with 150 μl of serumfrom an arthritic K/BxN mouse, or from a control (ctl) non-arthriticlittermate. Note the swelling and redness in the mouse injected witharthritic serum. Arthritis can be objectivated by measurement of anklethickness or clinical index assessement (right panels); the clinicalscore is defined as: 0, normal; 1, doubt; 2, two paws affected; 3, threepaws affected; 4, all limbs affected.

B: Disease induced by serum transfer is present in all recipients,whether transgenic or not, but is more intense in recipients whichpossess autoreactive T cells but cannot develop arthritis for lack of Bcells (K/BxN-μM^(o/o)).

C: Wane of arthritis, read out as clinical index, after a short courseof K/BxN serum administration (open dots), but persistence afterrepeated injection (black dots).

D: Arthritogenic activity of the serum IgG fraction. Left panel:RAG^(o/o) mice were injected with similar amounts (relative to startingvolumes) of serum from arthritic K/BxN mice (stars), the flow-through(crosses) or the fraction eluted from a protein-G column (closedcircles). Right panel: twice the amount of IgG from non-TCR tglittermates was incapable of inducing arthritis.

FIG. 2. Serum from K/BxN mice can transfer arthritis.

Knee sections from a typical K/BxN mouse (left) or a normal mouse 10days after two injections of 200 μl serum from an arthritic K/BxN animal(right). Note in both cases the thickened synovial lining, the massiveunderlying inflammatory infiltration, which extends over the cartilageand begins its destruction, and the presence of polymorphonuclear cellsin the articular cavity. Hematoxylin and eosin (H+E) staining, 10×objective.

FIG. 3. A 60 kD protein is the major target of K/BxN autoantibodies.

Total protein extracts (NP-40 extraction) from ankle (A), spleen (S) orkidney (K) were run on SDS-PAGE, transferred by electroblotting, andprobed with serum from a K/BxN arthritic mouse or a control littermate.The position of the prominent 60 kD band is indicated

FIG. 4. The 60 kD protein target of K/BxN serum is glucose-6-phosphateisomerase.

The 60 kD protein SEQ ID NO 1 from kidney extracts was immunopurified onimmunadsorbents made with K/BxN immunoglobulins. After digestion withtrypsin, peptides were purified by HPLC, and several sequenced byautomated Edman degredation. The three peptide sequences obtained belongto glucose-6-phosphate isomerase, and are underlined. Further confirmingthis identification, the molecular weight of a fourth peptide wasdetermined by mass spectrometry, and concides perfectly with the mass ofa tryptic fragment of GPI (wavy underline).

FIG. 5. Arthritic serum binds recombinant GPI.

A western blot was performed as in FIG. 3, except that the proteinloaded was recombinant GPI produced in E. Coli (as a fusion protein withGST). The blots were probed with serum from an arthritic K/BxN or acontrol mouse.

FIG. 6. ELISA assay detects anti-GPI antibodies in sera from arthriticmice, but not in controls.

In this enzyme-linked immunoassay, recombinant GPI, produced as above,was used to coat wells of ELISA plates. These were used to test foranti-GPI IgG in sera from arthritic or control mice. The values shownhere were obtained with serum dilutions of 1/16000 , but positivesignals could still be obtained with dilutions as great as 1/1,000,000 .The only negative arthritic mouse in the assay was an animal that hadbecome arthritic on the very day the serum was taken.

FIG. 7. Serum from K/BxN mice can transfer arthritis.

A: Recombinant GPI, or control recombinant protein—GST alone—, producedas above, was bound to a solid support, and used as an immunoadsorbentto remove anti-GPI antibodies from K/BxN serum. All the anti-GPI IgG wasin the bound fraction, as expected. The ability to transfer arthritiswas only found in the bound fraction, and eliminated from the fractionwhich flowed through the column.

B: A representative experiment, in which normal mice were injected withvarious fractions from the purification scheme shown in A. Arthritis wasevaluated and scored as above.

FIG. 8. Transfert of K/BxN serum can test therapeutic potential of amonoclonal antibody treatment.

Naïve 4 week old mice of the C57B1/6 strain were injected with 200 μl ofpooled serum from K/BxN mice on days 0 and 3. The mice were alsoinjected with test antibody BB5.1, which blocks the C5 complementfactor, at days −2, −1, +1, +5, and +8, or with vehicle only. The onsetof arthritis was followed by measuring ankles thickness in the followingdays. Mice treated with monoclonal antibody were protected fromarthritis, while those injected with vehicle only presented disease.

FIG. 9. Monoclonal anti-GPI antibodies induce arthritis when injectedtogether in naïve mice.

Naîve 4 week old mice of the Balb/c strain were injected with 1 mg ofpurified IgG from anti-GPI mAbs 6.121 and 2.99 dissolved inPhosphate-buffered Saline, or 1 mg of each, on days 0 and 3, the onsetof arthritis was followed by measuring ankle thickness in the followingdays. Mice treated with the two anti-GPI monoclonal antibodies incombination showed arthritis, but not those injected with either mAbalone.

EXAMPLE 1

Immunoglobulins Mediate KRN Arthritis

We have previously shown ((Kouskoff et al., 1996), U.S. Pat. No.5,675,060) that mice carrying all the genetic elements needed for theappearance of KRN arthritis, but deficient in B lymphocytes, are free ofdisease. To explore the possibility that arthritis development in theKRN model depends critically on a particular B cell product, we tried toprovoke disease in non-arthritic mice by transfer of serum from K/BxNmice. Severe joint swelling appeared in the animals injected with serumfrom arthritic K/BxN donors, but not in those receiving serum fromnon-arthritic BxN controls (FIG. 1A). Disease could be induced with aslittle as 100 μl of K/BxN serum, and showed up reproducibly. It wasprovoked very rapidly, measured by either clinical score or anklethickness, evident already within two days after serum injection.Arthritis could be obtained upon injecting sera from arthritic donorsinto normal mice, lymphocyte-deficient RAG^(o)/^(o) hosts, or B celldeficient K/BxN mice (K/BxN mice-μMT^(o/o)) (FIG. 1B). These resultsshow that serum components from K/BxN mice are sufficient to conferarthritis, although the more agressive disease seen with the latteranimals indicates that K/BxN T cells probably play an accessoryenhancing role in the effector phase.

The arthritis provoked by serum transfer presents all of thehistological features of the disease in regular K/BxN mice, includinginvasion of inflammatory cells, hyperplasia of synoviocytes, pannusformation, and cartilage destruction (FIGS. 2A and B).

The arthritis induced by serum injection is transient. In mice that hadreceived a single pair of injections, joint inflammation began tosubside after about 15 days (FIG. 1C); after 30 days, some of the jointsappeared quite normal, even in the animals that had initially been fullyarthritic. Disease transience could be overcome by repeated injectionsof serum from arthritic mice (FIG. 1C). That this was true even forRAG^(o/o) recipients suggests that instability of the serum compound isthe explanation for the transient nature of the disease.

The arthritogenic serum factor is a B cell-produced immunoglobulin (Ig):after fractionation of serum from K/BxN mice into IgG and non-IgGcomponents, only the IgG fraction is capable of provoking arthritis inRAG^(o)/^(o) hosts; its potency is similar to that of whole serum(relative to the starting volume) (FIG. 1D), as are the histologicalfeatures of the disease it induced (data not shown).

EXAMPLE 2

Molecular Target of the Pathogenic Immunoglobulins

Thus, an Ig produced by B lymphocytes is key to arthritis development inK/BxN mice. We then attempted to define their molecular targets. At theonset, one could have imagined that these could be antibodies directedagainst specific components of the joint, somehow generated by theinteraction of transgenic T cells reactive against the Ag7 molecule onthe surface of B cells; this could prevent the normal tolerance of Bcells towards self-components, or induce polyclonal B cell stimulationand the synthesis of Ig reactive against self (perhaps polymeric)components. We addressed this question:

1) By immunohistochemical analysis of RAGo/o mice after transfer of Igfrom K/BxN mice. These analyses showed deposition of transferred Ig notonly in the synovial tissue of the joint, but also in lining membranesof many other organs (spleen, kidney, liver, muscle; data not shown).

2) By Western blot analysis: Whole protein extracts were prepared fromthe ankle joint and from several other organs of RAG^(o/o) mice (toavoid the presence of Ig in the extract), electrophoresed on denaturingpoyacrylamide gels (SDS-PAGE), blotted, and probed with serum from K/BxNmice. Ig binding was revealed by probing with HRP-conjugated anti-mouseIgG. As can be seen on FIG. 3, a single dominant protein band could bedetected on these blots, at approximately 60 kD MW. This band was seenrepeatedly when sera from a number of K/BxN arthritic mice were used,but not with sera from control non arthritic littermates. Other proteinswere detected with some of the sera, but inconsistently and they werealways weaker than the 60 kD band.

We then attempted to identify this 60 kD protein. To this end, IgG froma large pool of K/BxN sera was purified by affinity chromatography onProteinG columns. This purified Ig (1.5 mg) was covalently bound toCNBr-activated Sepharose. This matrix was then used as animmunoadsorbant on which were loaded 50 mg of NP-40 extract from kidneyof RAG^(o/o) mice. The column was washed extensively, and bound proteinseluted at acid pH. They were analyzed by SDS-PAGE and Coomassiestaining. As expected from the results described above, a dominant bandof 60 kD was seen. This band was excised, the protein digested in thegel slice by Trypsin, as described in (Rosenfeld et al., 1992), and theresulting peptides resolved by reverse-phase HPLC. Several peptides wereidentified. Three of them were then sequenced by automated Edmandegradation on an Applied Biosystems 470A instrument. The sequences werecompared to public databases, using the BLAST program on the Swissprotdatabase. All three were found to belong to the proteinglucose-6-phosphate isomerase (aka phosphohexose-isomerase; EC 5.3.1.9;Swissprot database number P06745; sequence accession # 1230741;hereafter abbreviated as GPI). Their positions are shown in FIG. 4.Another of the peptides obtained was also found to belong to GPI, on thebasis of mass spectrometry analysis which fit exactly with the molecularweight predicted from the sequence (FIG. 4).

The molecular weight of GPI (62 636 kD) is very concordant with the MWof the target of K/BxN serum predicted from the Western blots, withinthe precision of MW estimation by PAGE.

GPI is an essential enzyme of the glycolytic pathway. It is an enzymeexpressed in essentially all tissues, whether normal or tumoral, withsome quantative variations, from the earliest stages of embryogenesis tolate in life of the animals (West et al., 1990; Hallbook et al., 1989;Warner et al., 1985). It is normally a cytoplasmic enzyme, but solubleGPI can be found in serum; it is found elevated in tumor patients(various organs), but not in a manner that make it a reliable marker(see for example (Neri et al., 1983; Schwemmer et al., 1985; Paulick etal., 1987; Gomm et al., 1988; Gurney et al., 1986)). GPI has also beenpurified independently as “Neuroleukin”, “Maturation factor”, or“Autocrine Motility Factor”, secreted factors of often limited potencyas a neurotrophic agent, or as agents promoting cell migration or tumorcell differentiation (Gurney et al., 1986; Faik et al., 1988; Niinaka etal., 1998; Xu et al., 1996). Genetic deficiencies in GPI result inhemolytic anemia syndromes (see for example (Kanno et al., 1996;Baronciani et al., 1996)).

To confirm that GPI is the protein recognized by the Igs present inK/BxN serum, we produced recombinant GPI in E. Coli. The coding sequenceof mouse GPI was amplified by Polymerase Chain Reaction, and the productcloned in the plasmid pGEx-4T-3 (Pharmacia). The recombinant protein, afusion product with Glutathione-S-transferase, was purified by affinitychromatography on Glutathione-Sepharose 4B column. The product wascharacterized on SDS-PAGE, and showed the expected size (data notshown). The gel was blotted, and strips were probed with sera from K/BxNor non-transgenic littermate controls. As can be seen in FIG. 5, allK/BxN sera reacted strongly, while control sera were negative,confirming that the GPI protein is the molecular target of anti-60 kDantibodies in K/BxN serum.

The recombinant protein was also used in Enzyme Linked ImmunoSorbentAssays (ELISA) to detect reactivity to GPI in sera of K/BxN mice ofdifferent ages. As previously described (Kouskoff et al., 1996), KRNarthritis appears at 28-32 days of age. As shown in FIG. 6, the assaydetected significant reactivity above background, up to dilutions of1/20 000 , in sera from K/BxN mice. No such reactivity was seen incontrol littermates. The assay can thus serve as a good diagnostic testof arthritis in these mice.

EXAMPLE 3

Anti-GPI Antibodies are the Pathogenic Immunoglobulins

Do anti-GPI antibodies constitute all the pathogenic specificities ofK/BxN serum, and can their removal eliminate the pathogenic potential ofthe serum. To address this question, we coupled 5 mg of recombinant GPIor of control GST protein, prepared as above, to CNBr-conjugatedSepharose. Pooled sera from K/BxN mice were applied sequentially tothese columns. The bound proteins were eluted at acid pH, and tested bytransfer into naive mice, along with an aliquot of the starting materialand of flow-through fractions. As shown in FIG. 7, all arthritogenicactivity was found in the fraction bound to the GPI-conjugated column,and none in the flow-through, even though the latter contained themajority of the immunoglobulin. These results demonstrate that anti-GPIantibodies are indeed the pathogenic Ig in serum from arthritic K/BxNmice, and that it can be adsorbed with recombinant GPI protein.

From these data, we conclude that anti-GPI antibodies produced in thetransgenic mice provoke arthritis. We have found (unpublished data) thatT cells expressing the KRN receptor are specifically stimulated byantigen-presenting-cells exposed to GPI antigen. These T cells in turnstimulate B cells producing anti-GPI immunoglobulin, which internalizeGPI effectively and thus receive help from T cells more readily thannon-specific B cells (Lanzavecchia, 1985). It is thus proposed thatself-reactive T cells against GPI or related circulating proteins,present in limited amounts in the circulation and thus unable to inducea paralyzing tolerance of the immune system, could induce similararthritogenic antibodies in human conditions such as RA.

EXAMPLE 4

The transfer of serum from arthritic K/BxN offers a model whereinpotential anti-arthritic formulations can be tested by administrationconcomitant with the arthritogenic serum or immunoglobulins.

As a proof of principle, we tested the ability of anti-C5 monoclonalantibody to interfere with arthritogenesis on the K/BxN model. We chosethis reagent because of prior evidence implicating complementcomponents, and in particular C5, in the generation of joint lesions inother mouse models of arthritis and in human RA patients (Watson et al,1987; Wang et al, 1995). Mice were injected with arthritogenic serumfrom K/BxN mice, and at the same time with the anti-C5 monoclonalantibody BB5.1, known to block C5 activity (Frei et al, 1987). As can beseen from FIG. 8, mice injected with the anti-C5 monoclonal wereprotected from disease.

EXAMPLE 5

If immunoglobulins present in the serum of K/BxN mice are able totransfer arthritis, it should be possible to isolate monoclonalantibodies (mAbs) also able to transfer disease to naïve recipients.Splenocytes from 30 and 50 days old K/BxN mice were fused according tostandard protocols for hybridoma derivation (de St Groth andScheidegger, 1980), and hybridomas selected in HAT medium in limitingdilution conditions in 96-well plates. Hybrids were screened forproduction of immunoglobulins reactive to GPI by testing culturesupernatants in an ELISA assay with recombinant GPI as a bound antigenand anti-mouse IgG as a developing reagent. Several positive wells wereselected for expansion, cloning by limiting dilution and testing forstability of the hybridoma lines. Eleven stable hybridoma linesproducing anti-GPI IgG were thus obtained (see Table 1 below).

TABLE 1 Anti-GPI monoclonal antibodies Monoclonal Antibody RecognizedPeptide Isotype 1.8 277-312 IgG1, κ 1.7  1-36 IgG1 1.11 47-82 IgG1 1.2424-59 IgG1, κ 2.99 conf. IgG1, κ 2.56 300-335 IgG2b, κ 2.67 277-312IgG2b, κ 6.149 277-312 IgG1, κ 6.121 277-312 IgG1, κ 6.65 conf. IgG1, λ6.96 277-312 IgG1, κ

The ability of these monoclonals to transfer disease was tested byProtein-G purification of mg amounts of the IgG produced by thesehybridomas, and intraveneous injection into naïve Balb/c recipients. Ascan be seen on FIG. 9, the coupled injection of anti-GPI immunoglobulinfrom the 6.121 and 2.99 hybridoma cells provoked arthritis in therecipients, but none of the antibodies were able to induce disease wheninjected alone. Among these antibodies, some works, such as the 2.99 and6.121 antibodies, while others don't. However, step e) of the processfor isolating antibodies capable of transferring arthritis according tothe invention allows to select the working ones.

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1. A method comprising injecting arthritic K/BxN transgenic mouse seruminto a mouse, wherein the injected mouse develops arthritis.